Sensitivity enhancement of paper-based sandwich immunosensor via nanobody immobilization instead of IgG antibody, taking aflatoxingenetic fungi as an analyte example

Aflatoxingenetic fungi producing highly toxic and carcinogenic mycotoxins and threatening human health widely exist in our living environment and food. Taking aflatoxingenetic fungi as an analyte example, here we report a way to improve the sensitivity of paper-based sandwich immunosensor for macromolecule. With a biomarker-specific nanobody and polyclonal antibody (IgG), two modes of paper-based immunosensors were constructed, immobilization nanobody mode and immobilization IgG mode. It was found that the sensitivity of the immobilization nanobody mode was at least 500 times higher than that of the immobilization IgG mode. The reasons for the large difference in sensitivity were also theoretically. Under the optimized conditions, the immobilization nanobody mode sensitivity reached 0.035 μg/mL aflatoxingenetic mycelia, each assay was finished within 15 min, and the spiked recoveries of aflatoxingenetic mycelium reference in peanuts were 77.0%~91.6%. And then, two important application cases were successfully demonstrated to identify the abundance of aflatoxingenetic fungi in market peanuts and peanut rhizosphere soil for environmental safety evaluation. This is the first report of time-resolved paper-based sandwich immunosensor for aflatoxingenetic fungi with nanobody immobilization, and the first sensitivity comparison between nanobody immobilization and IgG immobilization, which provides a new approach to develop highly sensitive immunosensor for macromolecular hazard materials. • The sensitivity of the immobilization nanobody mode was over 500 times higher than that of the immobilization IgG mode. • It provides a new approach to develop ultrahigh sensitive immunosensor for macromolecular hazard materials. • The developed paper-based sandwich immunosensor with LOD for aflatoxingenetic fungi reference was 0.035 μg/mL. • Evaluating risk of aflatoxingenetic fungi in food and environment.